CN103608006A - New combination comprising N-acetyl-L-cysteine and its use - Google Patents

Abstract

The present invention relates to a combination of N-acetyl-L-cysteine, selenium in the form of selenomethionine and melatonin, and a medical product or pharmaceutical composition comprising such combination,useful for the treatment of a variety of diseases and conditions, e.g. benign and malign neoplasia including various types of cancers, autoimmune diseases, neurodegenerative diseases, endocrinological diseases, type 2 diabetes, all types of fibrosis, amyloidosis, endometriosis, polycystic ovary syndrome, dysmenorrhea, dermatological diseases. The combination of N-acetyl-L-cysteine, seleniumin the form of selenomethionine and melatoninis also useful for cosmetic treatment of skin and as an antibacterial agent.

Description

Comprise new combination of ACETYLCYSTEINE and uses thereof

Invention field

The present invention relates to ACETYLCYSTEINE, selenium, be preferably the combination of selenomethionine (selenomethionine) and melatonin (melatonin), and the pharmaceutical composition that comprises this kind of combination, it is used for the treatment of various diseases and disease, optimum and the malignant tumor that for example comprises various cancer types forms (neoplasia), autoimmune disease, neurodegenerative diseases, endocrinopathy, type 2 diabetes mellitus, all types of fibrosiss, amyloidosis, endometriosis, polycystic ovarian syndrome, dysmenorrhea, dermatosis comprises vitiligo, alopecia and psoriasis.This combination is also as antibacterial and for cosmetic purpose.

Background technology

In born of the same parents, the control of oxidoreduction (redox) environment is relevant to cell processes basis with adjusting.For example, the cell cycle oxidoreduction more multi-oxidizer environment that causes out of hand, promotes to advance to the S phase also thereby cause abnormality proliferation from the G1 phase, and it is various neoplastic signs.And most of chronic inflammatory diseases also follow oxidoreduction out of hand.Towards being more oxidized the dysregulation of environment in born of the same parents thereby relevant to abnormality proliferation and inflammation and be therefore finally relevant to disease, described disease such as but not limited to, cancer, neurodegenerative diseases, diabetes, abnormal wound healing, fibrosis.In addition, in oxidation environment, protein can oxidizedly be modified, and conventionally forms aggregation after this process, is also the form of amyloplaste.

Outside stimulus is controlled the functional status of cell, affects function and the gene expression of key protein matter.By signal subspace, by sensing, also transduction is to the specific effect material (effectors) of being handled by protein post-translational modification, and wherein mercaptan (SH) reducing/oxidizing (redox) transforms performance basic role.Sensitivity cysteine residues in protein builds described oxidoreduction and transforms, and wherein " sensitivity " expression is derived from the electrochemical potential of lower cysteine/cystine redox couple (SH/S-S) of specific protein conformation.The mercaptan oxidation reduction conversion of synthesizing in (protein trafficking synthesis) and degraded control agent and cytoskeletal structure in for example enzyme, receptor, transporter, transcription factor, structural detail, protein transportation all affects whole cell homeostasis (cellular homeostasis) deeply.

In mammalian cell, the generation of oxidant derives from active oxygen and produces the elementary process such as glycolysis and mitochondrial respiratory chain finishing, and the most stable representative is therebetween hydroperoxides (H ₂o ₂).The oxidant producing must be under strict control and is needed the reverse of oxidative pathway.This oxidoreduction is controlled the impact that is subject to some low molecular weight substances and complicated enzyme network, comprising, glutathion reductase (GR) and selenium dependency glutathion peroxidase family (GPx).Reduced glutathion (GSH) is the dominant micromolecule amount thio-alcohol in most of organisms, many enzymes that its redox potential is responsible for oxidoreduction control in cell are used, comprise GR and GPx, with the redox state of recirculation they self.

Reduced glutathion (GSH) is with relative high concentration, even surpasses 10mM and is present in the tripeptides in everyone soma.It has many critical functions in vivo.As mentioned above, it can be regarded as main endogenous antioxidant, participates in: 1) control cellular redox state; And 2) control signal dependency protein, comprises the state of oxidation of outside stimulus receptor (for example hormone receptor).It brings into play basic role in multiple metabolism and biochemical reaction, and for example DNA is synthetic and reparation, protein synthesis, prostaglandin are synthetic, amino acid transport and enzyme activation.It also participates in regulating nitric oxide circulation, and many carcinogens and other exogenous material have important function for the optimal response in many immune system parts detoxify.Therefore, the interior most systems of body can be subject to the impact of glutathione system state.For example, glutathion defect state comprise HIV/AIDS, chemistry and infectious hepatitis, carcinoma of prostate and other cancer, cataract, Alzheimer, parkinson disease, chronic obstructive pulmonary disease, asthma, radiation poisoning, malnutrition, arduous body stress with aging (arduous physical stress and aging), and relevant to suboptimum immune response.Low glutathion also relates to strongly as cancer, AIDS, septicemia, wound, burn and training becoming thin and negative nitrogen balance excessively and seen in manic depression, major depressive disorder and the schizophrenia even.

Glutathione synthesis is from aminoacid Cys, Pidolidone and glycine.The sulfydryl of cysteine (mercaptan) group (SH) is as the biological activity of proton donor responsible glutathion.The supply of cysteine is the rate-limiting factor in cell synthesizing glutathion, because cysteine is relatively rare nutrient.Because suggestion in various diseases and disease supplements glutathion.

Yet the glutathion of orally ingestible can decompose and under one's belt already by gastrointestinal tract good absorption.Therefore by directly supplementing glutathion lifting glutathione level, be difficult.As the alternative supplement of glutathione precursor, be used to increase the plasma concentration of glutathion.ACETYLCYSTEINE (hereinafter NAC) is to compare the more simple molecule of glutathion, free diffusing be the glutathion precursor of bioavailable the most in nearly all tissue and cell.In several thiol reagents of its regulating cell redox state effect of test, NAC holds maximum mankind's prospect of the application.In the clinical use of NAC, related advantages is to have no side effect in fact.This compound can be used as mucolytic agent and the acetaminophen antidote after poisoning clinically for a long time.

Also approved that in recent years NAC has other beneficial property.For example, reported that NAC has antiinflammatory action and added to NSAID (non-steroidal anti-inflammatory drug) (NSAID) family.Shown before disclosure inventor in vitro in mammalian cell, NAC suppresses to breed and promotes reticent, (WO02/051405A1 further evolves in the differentiation of whole end, (the Cell Death and Differentiation (2005) such as T.Parasassi, 12 volumes, 10 phases, 1285-1296 page); E.K.Krasnowska etc. (Free Radicals Biology and Medicine2008,45 (11): 1566-72) and A.C.Gustafsson etc. (BMC Cancer (2005), 5:75).Found that NAC has significant anti-proliferative effect for cancerous cell and it has also been found that in film dystopy treatment in uterus effective at this point.In addition it has been used to treat polycystic ovarian syndrome (PCOS) and has been used for the treatment of various Other diseases and disease; for example, as nephroprotective agent, interstitial lung disease (interstitial lung disease), schizophrenia, manic depression and depression, and various other purposes have been proposed to be used in.

Therefore, NAC is effective for multiple use.Yet long-term NAC treatment causes NAC blood drug level to reduce, and therefore must increase risk (L Pendyala and P J Creaven, the Cancer Epidemiol Biomarkers Prev1995 of its valid density the undesirable side effect of corresponding increase; 4:245-251).For offsetting the attenuating effect of NAC, pulse therapy has been proposed can there is the removing phase.Another possible solution will be the concentration that reduces NAC available point, and it will be also favourable for short term therapy.

Recently for the two protection and/or therapeutical effect of selenium (hereinafter Se) and melatonin (hereinafter Mel), had broad interest.For example advised supplementing Se for prophylaxis of cancer, as antioxidant or immunostimulant.Studied cancer, immunological diseases and various other obstacle of being used for the treatment of of melatonin similarly.However, yet reported the dispute effect of these two kinds of materials.For example, admitted the antioxidant action of Mel, but it is also in the news sometimes as pro-oxidant (Cemeli E, Baumgartner A, Anderson D.Mutat Res.2009; 681:51-67;

caluba HC, the FEBS Lett.2001 such as Abuja PM; 502 (3): 127-31; The Toxicology and Applied Pharmacology2009 such as Radogna F; 239:37 – 45).

For Se, one group of large-scale experiment shows prevention (Lippman SM etc., the Journal of American Medical Association.2009 of carcinoma of prostate or other cancer of unrealized expection; 301 (1): 39-51).EAKlein (J Natl Cancer Inst.2009; 101:283-285) adopt warning conclusion: " complete good extensive controlled trial always do not verify we believe biology indicated and our model system be the imperfect appraisal of clinical effectiveness in real world." and; although reported that Se is for the preventive effect of diabetes risk and owing to the anti-oxidation characteristics of its " Insulin-Like " activity and selenoenzyme; a perspective study do not report any significant correlation between selenium and diabetes risk (Akbaraly TN etc., NutrMetab (Lond) .2010; 7:21).

Prior art

Balansky etc., " Interactions between N-acetylcysteine and sodium selenite in modulating the clastogenicity of urethane and2-acetylaminofluorene in mice ", Int.J.Cancer, 108 volumes, 2004,158-161 page, discloses and has been used in combination NAC and Se for weaken the side reaction of cytotoxic drug and chemopreventive agent in treatment of cancer.

Safarinejad etc., " Efficacy of selenium and/or N-acetyl-cysteine for improving semen parameters in infertile men:a double-blind; placebo controlled, randomized study ", J.Urol., 181 volumes, 1 phase, 2009, the 741-751 pages, network is disclosed in December, 2008, discloses combination or has used separately NAC and Se for improving semen quality in male infertility.Combination medicine-feeding NAC and Se cause additional beneficial effect.

deng, " Synergistic action of sodium selenite and N-acetylcysteine in acetaminophen-induced liver damage ", Hum.Exp.Toxicol., 27 volumes, 5 phases, 2008, the 425-429 pages, it is excessive to disclose combination or used separately NAC and Se to be used for the treatment of acetaminophen.Discovery NAC and Se combination are compared arbitrary independent reagent and are given better liver toxicity antagonism protection.

Emonet etc.; " Thiols and selenium:protective effect on human skin fibroblasts exposed to UVA radiation "; J.Photochem.Photobiol.; 40 volumes; 1 phase; 1997, the 84-90 pages, disclose and have been used in combination NAC and Se and with Cell protection, avoid the impact of UVA infringement.

Look etc., " Sodium selenite and N-acetylcysteine in antiretroviral-naive HIV-1-infected patients:a randomized; controlled pilot study ", J.Clin.Invest., 28 volumes, 5 phases, 1998,389-397 page, discloses combination oral administration NAC and Se with in order to improve cytometry and reduce the object of virus load in HIV patient.

Sener etc., " Melatonin and N-acetylcysteine have beneficial effects during hepatic ischemia and reperfusion ", Life Sciences, 72 volumes, 2003,2707-2718 page, discloses and has been used alone or in combination NAC and Mel with treatment hepatic ischemia.Find that Mel compares NAC more effective, and it is more effective to find that independent any compared in this combination of two kinds.

WO03/077900A1 and US2005/0164911A1 disclose by the method for administration NAC, melatonin or its combination prophylaxis of cancer or neurodegenerative diseases progress, and the medicine that comprises NAC and Mel.

WO00531376 has described and has comprised cysteine, selenium and melatonin together with the compositions of multiple other components.US20070231312, US2011027771, WO9833494 and US6207190 have described and have comprised other materials, and the different preparations of NAC, Se and Mel.US2004045566 has described the compositions that comprises glutathion, selenium and melatonin for absorbing harmful constituent from tobacco smoke in addition.None has described use selenomethionine described document.

In all documents of mentioning, selenium is used (selenium is used as such) with itself in the prior art.

Invention target

General target of the present invention is to provide the issue-resolution that increases NAC effect.The one side of this target is to provide medical product or medicine combination, and it provides ACETYLCYSTEINE (NAC) effect of increase.The one side of this target is to provide medical product or medicine combination, it provides the NAC of increase to be used for the treatment of for example disease in the mankind of mammal, for example, comprise that optimum and neoplasm, autoimmune disease, neurodegenerative diseases, endocrinopathy, type 2 diabetes mellitus, all types of fibrosis, amyloidosis, endometriosis, polycystic ovarian syndrome, dysmenorrhea, the dermatosis of various cancer types comprises the effect that vitiligo, alopecia and psoriasis and antibacterial infect.This target be to provide on the other hand medical product, its NAC that increase is provided is as antibacterial and for the effect of cosmetic purpose.

Summary of the invention

When inventor shows together with the selenium (Se) of NAC and selenomethionine form and melatonin (Mel) administration, the effect of ACETYLCYSTEINE (NAC) is enhanced.The strong NAC effect that increases of selenomethionine and Mel combination also also can reduce NAC concentration, thereby all improves its effect in short-term and long-term treatment.Test in vitro and in vivo after this combination treatment effect, adopt NAC, Se (for example form of selenomethionine, hereinafter SeMet) and Mel, inventor also shows to have avoided the ill effect of Se and Mel.

Therefore the combination that the invention provides NAC, SeMet and Mel is used for the treatment of various diseases and disease, for example, comprise that optimum and neoplasm, autoimmune disease, neurodegenerative diseases, endocrinopathy, diabetes, all types of fibrosis, amyloidosis, endometriosis, polycystic ovarian syndrome, dysmenorrhea, dermatosis and the antibacterial of various cancer types infects.This combination is also as antibacterial and for cosmetic purpose.In addition the invention provides the medical product and the pharmaceutical composition that comprise described combination, and comprise that simultaneously administration NAC, SeMet and Mel are to patient's Therapeutic Method.

The ACETYLCYSTEINE, selenomethionine and the melatonin that comprise separately or are together provided in one aspect of the invention, and/or the medical product of the upper acceptable salt of its physiology.Refer to that separately these three kinds of materials are parts of medical product, but can be used as independent unit, provide in the independent compartment of medical product.Refer to that together these three kinds of materials are alternately provided in the same section of medical treatment device, for example, as mixture.This medical product in one aspect of the invention in for simultaneously, order or separately combination medicine-feeding to mammal.

The component of medical product is for oral administration in one embodiment; For example ACETYLCYSTEINE component is the dosed administration with 5-45mg/kg/ days, and the selenium of selenomethionine form is to be the dosed administration with 0.02-0.08mg/kg/ days with the 0.4-1.2 μ dosed administration of g/kg/ days and melatonin.This medical product is to comprise ACETYLCYSTEINE, the selenium of selenomethionine form and the pharmaceutical composition of melatonin in one embodiment.

The ACETYLCYSTEINE, the selenium of selenomethionine form and the medical product of melatonin that comprise separately or are together for epithelium administration (epicutaneous administration) in another embodiment.In this medical product, ACETYLCYSTEINE is the concentration administration with 3-10wt-% in one embodiment, and the selenium of selenomethionine form is to be the concentration administration with 0.01-0.2wt-% with the concentration administration of 0.3-1wt-% and melatonin.This medical product is to comprise ACETYLCYSTEINE, the selenium of selenomethionine form and the pharmaceutical composition of melatonin in one embodiment.

This medical product is to be used for the treatment of optimum or malignant tumor formation in one embodiment, comprises various cancer types.It is process and be used for the treatment of dermatosis for beautifying skin in another embodiment.This medical product also can be used to treat autoimmune disease, neurodegenerative diseases, endocrinopathy, type 2 diabetes mellitus, all types of fibrosis, amyloidosis, endometriosis, polycystic ovarian syndrome, dysmenorrhea and Other diseases.This medical product is as antibacterial in one embodiment.

Comprise ACETYLCYSTEINE, the selenium of selenomethionine form and the medical product of melatonin and in the present invention be on the other hand as antibacterial, for together with (together with) or on the surface of the medical treatment device that is applicable to contact with body fluid.This medical product for example can be used in the coating of this kind of medical treatment device, maybe can be incorporated to this kind of medical treatment device material, for example, in plastics.In one embodiment of the present invention, the selenium of ACETYLCYSTEINE, selenomethionine form and melatonin are contained in the hydrogel coating for medical treatment device.This medical product as antibacterial can further comprise additional activity compound for example germ killing drugs, precious metal solution and/or dexamethasone.

The invention provides in one aspect the ACETYLCYSTEINE comprising separately or together, selenium and the melatonin of selenomethionine form, and/or the antibacterial of the upper acceptable salt of its physiology.This antibacterial is the hydrogel coating being used in for medical treatment device in one embodiment.It is for the plastic concordancy with medical treatment device (integration) in another embodiment.

The invention provides on the other hand the medical treatment device that comprises medical product; selenium and melatonin that this medical product contains ACETYLCYSTEINE, selenomethionine form, and/or the upper acceptable salt of its physiology and optional additional activity compound for example germ killing drugs, precious metal solution and/or dexamethasone.

The invention provides on the one hand the ACETYLCYSTEINE comprising separately or together, selenium and the melatonin of selenomethionine form; and/or the purposes of the medical product of the upper acceptable salt of its physiology, be used for the treatment of and comprise that the optimum of various cancer types and malignant tumor formation, autoimmune disease, neurodegenerative diseases, endocrinopathy, type 2 diabetes mellitus, all types of fibrosis, amyloidosis, endometriosis, polycystic ovarian syndrome, dysmenorrhea, dermatosis comprise that vitiligo, alopecia or psoriasis or antibacterial infect.The present invention also provides medical product for beautifying skin, to process and as the purposes of the antibacterial together with the medical treatment device that is applicable to contact with body fluid.

The invention provides on the one hand methods for the treatment of diseases; or for cosmetic purpose; comprise selenium and the melatonin of combination medicine-feeding ACETYLCYSTEINE, selenomethionine form simultaneously, sequentially or separately, and/or the upper acceptable salt of its physiology, to the mankind or mammalian subject.This disease or cosmetic purpose are to be selected to comprise that the optimum of various cancer types and malignant tumor formation, autoimmune disease, neurodegenerative diseases, endocrinopathy, type 2 diabetes mellitus, all types of fibrosis, amyloidosis, endometriosis, polycystic ovarian syndrome, dysmenorrhea, dermatosis comprise that vitiligo, alopecia or psoriasis or antibacterial infect, or beautifying skin is processed.

Accompanying drawing summary

In following description, with reference to accompanying drawing, will explain in more detail the present invention, wherein:

Fig. 1 has shown by glutathion (GSH) metabolic cycles of SeGSHpx (cGPx (Se)) and glutathion reductase (GR).GR activity needs NADPH, and it is provided by enzymatic glucose-6-phosphate dehydrogenase (G6PDH) activity.G6P: G-6-P salt; 6PG:6-phosphogluconate; RO: alkoxy free group; OH: hydroxy radical; ROOH: hydroperoxides.

Fig. 2 has shown and has adopted as described in Example 1 the NAC+SeMet+Mel treatment effect for patients with vitiligo in three months.

Fig. 3 A has shown and has adopted as described in Example 2 a NAC+SeMet+Mel treatment effect for skin texture in month.Silicone rubber skin copy MIcrosope image shows the fine rule network that characterizes the micro-looks of skin surface.Notice more regular network after treatment.Amplification: 5X or 10X, as reported in figure hurdle.

Fig. 3 B has shown the fast fourier transform (FFT) of image in some Fig. 3 A, shows the irregular pattern (A and B) of general single direction in the front sample for the treatment of, and after treatment, observes regular circular pattern (C and D).

Fig. 4 has shown the result from test described in embodiment 3.As indication, adopt 0.2mM menadione and/or 0.4mM NAC and/or 0.2mM SeMet and/or 2mM Mel processing K562 cell.In this block diagram, symbol Se refers to SeMet all the time.

Fig. 5 has shown the result from test described in embodiment 3.As indication, adopt 0.133mM menadione and/or 0.4mM NAC and/or 0.2mM SeMet and/or 2mM Mel processing HL60 cell.In this block diagram, symbol Se refers to SeMet all the time.

Fig. 6 has shown the result from test described in embodiment 3.As indication, adopt 0.5mM menadione and/or 1mM NAC and/or 0.2mM SeMet and/or 1mM Mel processing HL60 cell.In this block diagram, symbol Se refers to SeMet all the time.

Fig. 7 has shown the result from test described in embodiment 3.As indication, adopt 0.133mM menadione, 0.2mM SeMet, 2mM Mel and different N AC concentration to process HL60 cell.

Fig. 8 has shown the result from test described in embodiment 4, confirms antiproliferative and the differentiation of NAC and SeMet and Mel combination.Amplified gene P21 inhibitor (average 5 indexes) and differentiation marker thing SI (5 indexes), ALPI (4 indexes), the average multiple of CLDN1 (6 indexes) and PCDH1 (5 indexes) changes.Significance, p<0.05, gets rid of two samples marking (*), wherein the combination of NAC+Se and NAC+Mel and NAC+SeMet+Mel there was no significant difference.In this block diagram, symbol Se refers to SeMet all the time.

Fig. 9 has shown the effect contrast of independent NAC and three kinds of combinations of substances.In this block diagram, symbol Se refers to SeMet all the time.

Detailed Description Of The Invention

NAC

ACETYLCYSTEINE (NAC) is known low-molecular-weight drug, has chemical formula

For the purposes of the present invention, NAC can also its dimerization form (di-NAC) administration.

Found that NAC is by various mechanism performance molecules and physiological effect.The feature of NAC relates generally to its mercapto, and it makes it in most of bio-chemical pathways of glutathion (GSH) effect is effective.NAC is treated to Cys by cell and is used in the de novo synthesis of GSH, is therefore considered to the precursor of GSH.

Although the details of the NAC mechanism of action is not also understood completely, certainly they are inevitable owing to its mercapto.Itself thus relate in cell complicated mercapto redox cycle, and thereby affect the adjusting of cellular redox state and born of the same parents are interior and intracellular signal.In this redox cycle, some enzymes play a role, and the generation that its oxidoreduction changes is by the oxidation/reduction of glutathion (GSH).Have NAC and get involved the formerly evidence in this figure, as summed up in Fig. 1, NAC has similar or even higher effect than GSH.In this respect, NAC can be considered to effective antioxidant.

On physiology, very importantly disulphide forms and the circulation of cracking, and this is a kind of general mechanism that regulates protein active and cellular signal transduction.Enzyme, for example Protein-tyrosine-phosphatase and tyrosine kinase, for example, play a part crucially in controlling cell cycle, cell proliferation and differentiation, and the redox state of many these enzymes by its cysteine regulates.

NAC once and was at present still widely used as mucolytic agent, and its model of action is conventionally owing to the oxidoreduction cracking of the responsive cysteine disulfide bridge bond in mucus egg.In addition NAC has been the poisoning main therapy of acetaminophen.

NAC has also been identified and has had other beneficial property in recent years.For example reported that NAC has antiinflammatory action, this is also one of reason being added NSAID (non-steroidal anti-inflammatory drug) (NSAID) family.Further find that NAC inhibition cell proliferation also promotes reticent, further in last differentiation, evolving eventually.Found that NAC has significant anti-proliferative effect for cancerous cell and it has also been found that in its film dystopy treatment in uterus effective at this point.For example, the relative recent findings of inventor NAC for epithelial origin cancerous cell, there are significant anti-proliferative effect (Cell Death and Differentiation2005,12 (10): 1285-1296; BMC Cancer2005,5:75).

In cancer research, the anti-proliferative effect of NAC and cell death or toxicity are irrelevant, but because the activation of physiology's differentiation pathway brings, it can be considered to be cell function towards the normalization of tissue of origin.

Related advantages during NAC is used is to have no side effect in fact.Shortcoming is that its effect in long-term treatment reduces, and report blood drug level reduces so that required dosage increases, thereby has also increased the risk of some adverse events.

NAC and Se and Mel combination

On this basis, inventor's object is that by use simultaneously, can bring into play collaborative other medicines with NAC reduces effective NAC dosage.The minimizing of NAC effective dose is simultaneously for short-term and long-term treatment.For this object focused attention is to the enzyme network of controlling mercaptan oxidation reduction circulation, particularly those reactions relate to those of glutathion recirculation by the enzyme such as GSH-Px(glutathione peroxidase) (GPx) and glutathion reductase (GR).Concentrate on these redox reaction streams and be proved to be correct, by observing them, account for 99.9% (the DP Jones.Am.J.Physiol.Cell Physiol.2008 nearly of redox reaction biology; 295:C849-C868) and NAC effect should be mainly relevant to the modification of these polyenzyme activity (Costa G, waits The Scientific World JOURNAL2010 for Parasassi T, Brunelli R; 10:1192 – 1202).

Reported that the GR adopting after NAC long-term treatment expresses minimizing (L Pendyala and PJ Creaven, Cancer Epidemiol Biomarkers Prev1995; 4:245-251).Also exist report to show that supplementary Mel can obtain another relevant enzyme in GR and mercaptan oxidation reduction reaction: GSH-Px(glutathione peroxidase) (GPx) is expressed and/or active increase (Hardeland R, Cardinali DP, the ProgNeurobiol.2011 such as Srinivasan V; 93 (3): 350-84, open day of network: on December 28th, 2010; Garc í a T, Esparza JL, Giralt M etc., Biol Trace Elem Res.2010; 135:220-32).

In this figure, Se represents the cofactor of some glutathion peroxidase (GPx).Se is necessary (referring to such as Free RadicBiol Med.2007 such as Bulato for GPx activity; 42:118-23), and in the situation that not there is not GPx enzyme, do not bring into play any specific antioxidation.

Inventor so inference do not have harmful effect for guaranteeing balanced action, and Se is necessary for the form of SeMet administration together with Mel, at GR with GPx is upset and GPx can bring into play under its active condition completely.For this reason, NAC can guarantee the reduction potential carrying out for integral power, at this, finally compares glutathion (GSH) more effective.

Biochemical based on mercaptan oxidation restoring system, inventor is intended to reduce in NAC treatment concentration situation to be increased the effect of NAC and in long-term treatment, guarantees NAC effect.The realization of this object is to be present in the key element in mercaptan oxidation restoring system by stimulation.

For: 1) recover/strengthen to be responsible for expression/activity that mercaptan signal conducts the enzyme of oxidoreduction control; 2) recover suitable NAC effect, even in long-term treatment; And 3) reduce effective NAC concentration, NAC treatment supplements combined with selenium and the melatonin of selenomethionine form.This combination inspires and to need selenium from GPx activity, and the effect of the melatonin of reporting in the expression/activity that increases GR and possible GPx.

Therefore, the present invention is based on strengthen the object of NAC effect by strengthening mercaptan oxidation restoring system, this enhancing be by:

1) by supplementing the Se of selenomethionine (SeMet) form, stimulate the activity of GSH-Px(glutathione peroxidase) (GPx), SeMet is the bioavailable form (bioavailable form) of Se.

2) use selenomethionine to replace selenium very important for this effect.[2) by Mel, increase expression and/or the activity of glutathion reductase (GR) and GSH-Px(glutathione peroxidase) (GPx).

Antioxidation, antiproliferative and differentiation effect by test NAC have confirmed the NAC effect of combining with these two kinds of added substances.Combined effect between the Se of NAC, Mel and SeMet form and possible collaborative test are by using in vitro two kinds to face the cell line (embodiment 3 and 4) that oxidation stimulates, and in patients with vitiligo body and in dermatological studies (embodiment 1 and 2).

Antiopxidant effect about NAC in mercaptan oxidation restoring system, stimulates the minimizing generating to measure by hydroperoxides, finds:

1) by this association, clearly strengthened NAC effect.In some situations, compare with the NAC of SeMet and Mel combination himself almost more effective three times.

2) NAC concentration can reduce, and uses the concentration that is less than half to reach similar effect.

3) unexpectedly,, when only combining two kinds of materials, when NAC+SeMet or NAC+Mel, in some cell systems, observed disadvantageous oxidation effect.This is beat all, because report that in scientific literature these combinations are favourable (Safarinejad MR.J Urol.2009; 181:741-51.

deng Hum ExpToxicol.2008; 27:425-9).On the contrary, the combination of observing three kinds of materials has recovered cytoprotective, has the final effect of almost completely free oxidation effect.

About the relevant effect that reduces cell proliferation and induce atomization of NAC, find:

1), than independent use NAC, by combining with SeMet and Mel, the antiproliferative effect of NAC doubles.

2) differentiation of NAC has clearly strengthened by above combination, according to special markers, than independent use NAC, has improved 30-50%.

3) by analyzing some specific differentiation marker things, the combination of observing NAC+SeMet or NAC+Mel has the effect that equals to three kinds of combinations of substances.Yet the trend of knowing when three kinds of combinations of substances are used with the whole increase of NAC effect.

Administration

The new combination of the Se of NAC of the present invention, SeMet form and Mel can be simultaneously, order or separately combination medicine-feeding to the mammal mankind for example.Combination medicine-feeding refers to that all three kinds of materials are administered to mammal together at same time point simultaneously.Sequential combination administration refers to that these three kinds of materials are administered to mammal in the identical course for the treatment of, for example, during identical treatment day, treatment week or the treatment moon, so that all substances combination is present in mammal, this material is by administration, and in setting order, different time points is given to mammal.Separately combination medicine-feeding refers to that these three kinds of materials are administered to mammal so that all substances combination is present in mammal in the identical course for the treatment of equally, but this material is by administration, and in irregular order, different time points is given to mammal.Administration can be (intestinal or parenteral) local or system.Typical case's administering mode comprises oral administration and epithelium administration.Other possible administering mode comprises in intravenous, Intradermal, subcutaneous, intramuscular, intravaginal, intrauterine, intraperitoneal, rectum, nose, sheath, suction and intravesical administration.

Preparation

According to the combination of NAC of the present invention, SeMet and Mel, can be drug alone preparation/compositions by administration, only comprise separately a kind of in three kinds of active component, comprise respectively NAC or SeMet or Mel.Or this combination can be by administration in comprising the pharmaceutical composition of two kinds or all three kinds of active component.In arbitrary situation, comprising only pharmaceutical composition a kind of or NAC and/or SeMet and/or Mel combination can the known mode of drug world technical staff itself be produced.

This pharmaceutical composition for example can be suitable for oral administration.Such composition can multi-form administration, is preferably at present tablet.Other form, such as capsule, suppository, solution, suspension, syrup etc. also can be expected.Pharmaceutical preparation at the dosage unit form for oral administration can mix one or more active component and the solid, powder carrier or the excipient that are used as carrier for active principle or medium, for example lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose derivative, gelatin, citric acid, sodium citrate, carbonic acid (acid) sodium or in addition suitable carrier; The hydroxide of stable material such as alkali compounds such as carbonate, sodium, potassium, calcium, magnesium etc. and oxide and lubricant be magnesium stearate, calcium stearate, sodium stearyl fumarate and polyethylene glycol wax for example.Then mixture is processed into granule or compacting in flakes.It is available as long as dosage form is still avoided enteric coating coated particle and the tablet of acid catalysis degraded under one's belt with regard to prolection compound.This enteric coating is selected from pharmaceutically acceptable enteric-coating material such as Cera Flava, Lac or anion film forming polymer etc. (if preferred and suitable plasticizer combinations).Can in coating, add various dyestuffs to distinguish tablet or the granule with different activities compound amount.

Can adopt the mixture that comprises reactive compound, vegetable oil, fat or the suitable medium of other soft capsule to prepare soft capsule.Soft capsule also can be as mentioned above by enteric coating.

Hard capsule can comprise active ingredient composition granule or enteric coating granule.Hard capsule also can comprise and the pressed powder carrier reactive compound that for example lactose, sucrose, sorbitol, mannitol, potato starch, amylopectin, cellulose derivative or gelatin combine.This capsule also can be as mentioned above by enteric coating.

One option is to provide NAC and/or SeMet and/or Mel in slow releasing preparation (being also expressed as sustained release or controlled release).By reducing active substance, enter diffusion and the absorption rate of blood flow, this kind of preparation can longer interval administration larger dose.This dosage is distributed in a small amount in blood in long-time subsequently, for example in the every day therapeutic scheme situation of twice through 12+12 hour.Much different slow release technique and preparation be in prior art, know already and can be applicable to the present invention.In this type of technique, active substance is for example encapsulated in the coating or substrate of the insoluble or less dissolving in the body fluid place of administration position.The preparation with slow release stomach function regulating protection combined effect is also fine and is used in the present invention.

Liquid preparation for oral administration can be prepared as syrup or suspension form, for example, comprise 0.2% to 20% weight active component and remainder by solution or the suspension of sugar or sugar alcohol and ethanol, water, glycerol, propylene glycol and/or Polyethylene Glycol compositions of mixtures.If desired, this type of liquid preparation can comprise coloring agent, flavoring agent, glucide and carboxymethyl cellulose or other thickening agent.Liquid preparation for oral administration also can be prepared as dry powder form to adopt before use appropriate solvent to rebuild.

For topical, NAC and/or SeMet and/or Mel can respective pure form be applied, as liquid.More preferably they can be by administration in the compositions/preparation that comprises dermatological acceptable carrier, and it can be solid or liquid, is emulsifiable paste, ointment, lotion, liniment, powder etc.The example of liquid-carrier comprises water, alcohol or glycol or water-ol/diol mixture, and the compound wherein existing is can effect level dissolved or disperse, optionally by means of nontoxic surfactants.Can add thickening agent such as synthetic polymer, fatty acid, soap and ester, fatty alcohol, modified cellulose or modified mineral material to form ointment, lotion, paste, gel etc.The example of solid carrier comprises finely-divided solid such as Talcum, clay, microcrystalline Cellulose, silicon dioxide, aluminium oxide etc.Also can add auxiliary agent for example aromatic and additional antibacterial.Galenic (galenic) preparation for topical comprises water, ceteareth 25 tristerins, triethanolamine stearate, cetearyl alcohol, caprylic/capric triglyceride, simethicone, hydrogenating glycerine polyisobutylene, polysorbate 60, pentanediol and optional Rhizoma et radix valerianae in a preferred embodiment.

For intravenous or intraperitoneal administration, active compounds solution can be prepared in water, optionally mixes with nontoxic surfactants.The preparation of dispersion liquid also can, in glycerol, liquid macrogol, glyceryl triacetate and composition thereof and oil, optionally be encapsulated in liposome.That final dosage form should be is aseptic, fluid and manufacture and storage requirement under stable.Liquid-carrier or medium can be solvent or liquid dispersion medium, for example comprise, water, alcohol, polyhydric alcohol (for example, glycerol, propylene glycol, liquid polyethylene glycol etc.), vegetable oil, nontoxic glyceryl ester and suitable mixture thereof.Maintaining of adequate liquidity can be for example, by forming liposome, by safeguarding the particle diameter that needs or by using surfactant in dispersion liquid situation.Can pass through various antibacterial and antifungal, for example, the prophylaxis of microbial effects such as p-Hydroxybenzoate, methaform, phenol, sorbic acid, thimerosal.To preferably comprise isotonic agent in many cases, for example, sugar, buffer agent or sodium chloride.Can be by use delayed absorption agent in compositions, for example aluminum monostearate and gelatin, the long-term absorption that brings Injectable composition.The preparation of aseptic injectable solution is by be incorporated to the reactive compound of demand, then filtration sterilization according to the rules together with above-named various other compositions in suitable solvent.As for the sterilized powder for the preparation of sterile injectable solution, preferred preparation method is vacuum drying and Freeze Drying Technique, the powder of the arbitrarily additional required composition before its acquisition active component adds and is present in aseptic filtered soln.

For the dosage unit of rectally, can be prepared as the suppository form that contains the active substance mixing with neutral fat substrate or they can be prepared as gelatin rectal capsule form, be included in there is vegetable oil, paraffin oil or can be prepared as the form of the dry micro-enema preparation that administration rebuilds before in appropriate solvent for the active substance of the mixture of the suitable medium of gelatin rectal capsule or form that they can be prepared as ready-made micro-enema or they.

Solution for parenteral can be prepared as the pharmaceutically solution of acceptable solvent of active component of the present invention, and preferred concentration is 0.1-10% weight.These solution also can comprise stabilizing agent and/or buffer agent and can be manufactured in different unit dose ampullas or liquid medicine bottle.Solution for parenteral also can be prepared as the dry agent of using at any time front employing appropriate solvent to rebuild.

For NAC, the present invention needs the drug quality of Due Diligence NAC preparation to obtain effective dose.NAC is not stable molecule, and its active mercaptan residue can be oxidized easily by oxygen, illumination or other radiation, to such an extent as to cannot realize effective dose.Therefore preparation preferably keeps in Dark Place.For oral administration, preferably NAC to be prepared in having the fuse of sodium bicarbonate, it helps part during stripping to remove the oxygen from water.

The NAC that has observed high dose can cause stomachache.For overcoming this, an option is that NAC is provided in having the preparation of stomach protection, and it is suitable for preventing that NAC from discharging/dissolving at stomach.This type of preparation is commonly known in the art and can uses in company with the present invention.For example can use opposing gastric juice and can after it is by stomach, only in intestinal, discharge the tablet coating of medicine.General preparation comprises polymer for example cellulose derivative, methacrylate amino ester copolymer.This coating is by using pH sensitive polymer protection label to avoid decomposing in stomach acidity environment, and this pH sensitive polymer responds the increase of pH after by stomach, swelling or dissolve to discharge medicine.

Dosage

For being administered orally to the mankind and other mammal, the suitable daily dose of NAC is approximately 5～45mg/kg/ days, preferably 20～30mg/kg/ days.The suitable daily dose of SeMet is about 0.0004～0.0012mg/kg/ days, preferred about 0.0008mg/kg/ days, and the suitable daily dose of Mel is about 0.02～0.08mg/kg/ days, preferred about 0.04mg/kg/ days.Can be once a day or be divided into twice or more of this dosage, preferably three or four times, administration every day is one or two dosage (for example pill) separately, and wherein each dosage can comprise for example 0.15-3.0g NAC, and preferably 0.3-0.6g NAC is also preferably three times on the one; 0.028-0.110mg SeMet or preferred about 0.055mg SeMet; And 1-6mg Mel or preferred about 3mg Mel.

For oral administration, the daily dose weight ratio of NAC and SeMet in the scope of 4000:1-60000:1, preferably in the scope of 5000:1-40000:1 or 25000:1-37000:1,32500:1 most preferably.The daily dose weight ratio of NAC and Mel in the scope of 60:1-2000:1, preferably 150:1-1000:1 or 500:1-750:1, most preferably 600:1.Typical day dosage weight ratio of three kinds of materials is that NAC:SeMet:Mel is about 32500:1:54.Described material can be with described ratio while, independent or administration in the compositions that comprises all three kinds of materials.

For epithelium administration (for example, by emulsifiable paste, ointment, lotion, liniment or analog), the debita spissitudo of NAC is about 3-10wt-%, preferably 5wt-%.The debita spissitudo of SeMet is about 0.3-1wt-%, and preferably 0.5wt-%, and the suitable daily dose of Mel is about 0.01-0.2wt-%, preferably 0.1wt-%.This emulsifiable paste, ointment or analog can once-a-day administration or can be used every day twice or repeatedly, preferably administration every day three or four times.

For epithelium administration, NAC and weight-% of SeMet compare in the scope of 3:1-33:1, preferred about 10:1.NAC and weight-% of Mel compare in the scope of 15:1-1000:1, preferred about 50:1.The typical weight of three kinds of material epitheliums administration-% ratio is that NAC:SeMet:Mel is about 50:1:5.Described material can be with described ratio while, independent or administration in the compositions that comprises all three kinds of materials.

Depend on treated disease or disease, sustainable one day for the treatment of cycle or a couple of days are until the several months.

For the longer treatment cycle that adopts NAC and SeMet and Mel, for example 1-2 month or more, treatment can be intermittent.Intermittent administration or treatment refer to that treatment interrupts on the cycle, i.e. this pharmaceutical composition a period of time of administration, and a couple of days for example, interruption of the administration then, administration medicine compositions a period of time not wherein, a couple of days for example, and then follow administration.Intermittent treatment can be regular, for example, treat fixing natural law or all numbers, then interrupts fixing natural law or all numbers.Example comprises that employing weekly treatment interrupts 3 days or treat within 2 weeks, interrupting afterwards the iteration scheme of 1 week for 4 days afterwards.Regularly a kind of special circumstances of intermittent treatment are pulse therapies, continue regularly treatment and interrupt, and interrupt two days later two days etc. such as every other day administration or administration.The irregular intermittent therapeutic scheme that irregularly repeats or have a more complicated plan also can be expected, for example, depend on the response to treatment.In the different illustrative embodiments of the present invention, the prescribed dose of NAC and SeMet and Mel is that administration 3-5 interrupts 2-4 day after the Consecutive Days, or administration 1-3 interrupts 1-2 day after the Consecutive Days.

The preferred dose of NAC, SeMet and Mel by depend on selected particular formulations, route of administration, the disease of controlling or situation character and patient's body weight, age, situation and species (mankind or animal).

When NAC is during together with the SeMet of optimal dose and Mel administration, the known preferred dose of NAC treatment specified disease or situation can typically approximately reduce by half.As an example, the preferred individually dosed dosage that is used for the treatment of endometriotic NAC is 20-90mg/kg/ days, preferred 30-60mg/kg/ days, oral administration 2 months or more of a specified duration.When NAC and SeMet and Mel combination medicine-feeding, NAC dosage can be reduced to 15-30mg/kg/ days, administration 2 months or more of a specified duration.

Purposes/medicine indication of the present invention

The present invention who comprises NAC, the Se of selenomethionine form and the combination of Mel can be used to wherein NAC and can be used or known any object with effect, for example glutathion participation therein and/or wherein mercaptan (SH) reducing/oxidizing (redox) conversion performance basic role, as in signal conduction situation, also by hormone, order about, or wherein can utilize the antiproliferative of NAC to urge in the disease of differentiation effect, antioxidation or anti-inflammatory effect.It for example can be used to treatment and comprise the optimum of various cancer types and malignant tumor formation, type 2 diabetes mellitus, various types of fibrosis, endometriosis, polycystic ovarian syndrome, dysmenorrhea and dermatosis, comprises vitiligo and psoriasis.Because oxidative phenomena and the dependency that forms amyloplaste in protein aggregation, the present invention can be used to prevention and treat various types of amyloidosis, for example Alzheimer.In addition it can be used as antibacterial and for cosmetic purpose.Other purposes also comprise treatment HIV/AIDS, chemistry and infectious hepatitis, cataract, parkinson disease, chronic obstructive pulmonary disease, asthma, radiation poisoning, malnutrition, arduous body stress, aging, septicemia, wound, burn, manic depression, major depressive disorder and schizophrenia, and for thering is the immunoreactive patient of suboptimum.

As the example for the treatment of skin disease, the extreme that adopts these patients with vitiligo of three kinds of combinations of substances treatment and observe decolouring in treatment after three months reduces (extreme reduction), and (embodiment 1, Fig. 2).

As the example of cosmetic treatments, monitoring adopts the galenical treatment facial skin experimenter's of month of containing these three kinds of compounds skin texture.The upper thread performance of cream preparation therapeutic alliance that employing contains NAC, SeMet and Mel one Ge Yuehou， face is more regular, and cutaneous manifestations is more compacted and be smooth and skin is irregular and pimple (pimple) minimizing.

NAC, SeMet and Mel combination are as antibacterial

The combination that the inventor also shows (embodiment 5) NAC, SeMet and Mel is effectively as antibacterial, for example, in the time of in being contained in such as the medical apparatus and instruments of latex or polyvinyl chloride conduit or the coating on equipment.Or the combination of NAC, SeMet and Mel can be incorporated in the plastic material for medical treatment device.

For example, hydrogel coating can be used as the method for solid matrix surface modification.The example of hydrogel coating comprises the water-insoluble hydrogel based on polyurethane (PUR) or polyvinylpyrrolidone (PVP).Except as improved Biocompatibility, hydrophilic and lubricated advantage, hydrogel coating has also brought that for example NAC, SeMet and Mel are incorporated to the additional probability on surface by activating agent.The hydrophobic drug compositions of combining hydrophilic substrate and NAC, SeMet and Mel looks like promising especially.

Hydrogel modified surface has the attendant advantages on unmodified surface, because it can be used as the drug depot for localized drug delivery.For example, the drug dose time should continue to few a few hours under specific circumstances, but is no more than 3 days.For example, in the implanting device situation as tracheotomy tube, when anti-inflammatory activity material should discharge to prevent at the beginning the follow-up side effect as tracheal stenosis, but not interfere with subsequent agglutination and the epithelium normal cell division in forming afterwards, it can be satisfies the demand.By using hydrophilic hydrogel substrate can obtain easily this releasing properties together with hydrophobicity NAC, SeMet and Mel, this is because of hydrophilic matrix characteristic as controlled drug delivery system during its swelling.

Except NAC, SeMet and Mel, can in the coating together with medical apparatus and instruments and equipment use or plastics, add other active substance, for example germ killing drugs, precious metal solution and/or dexamethasone.

Activating agent, be NAC, SeMet and Mel, and one or more possible additional activity materials can be usingd two kinds of different modes and are incorporated to coating: I. is as solution composition in coating formation step arbitrarily, and for example, between hydrogel coating polymerization period, or II. passes through dipping bath.Additional step after the latter's mode II. preferably forms as hydrogel coating, but it completes also and can there is no this coating, and directly to medical treatment device.Mode II. correctability, as the equipment of silica gel catheter or polyglycolic acid absorbable suture, does not have polymer coating step or add active substance after polymer coating step.

Inventor has confirmed the effect that activating agent is incorporated to, by testing drug be dissolved to have 20% methanol or ethanol phosphate buffer (PBS), extract, and if germ killing drugs, by through suppressing growth district method (data are unlisted).Studied medicine dissolution rate and load capacity, and the dependence of coating stability and technological parameter.Inventor observes NAC, SeMet and the spreading effect of Mel during swelling behavior, because the solid particle of material (NAC, SeMet, Mel) is settled out coating solution extremely around.

The inventor show (embodiment 5) than without coating framework material and do not have NAC, SeMet and Mel hydrogel coating material the two, at the upper hydrogel coating that comprises NAC, SeMet and Mel that uses of tube material (polrvinyl chloride, latex), significantly reduced bacterial growth on this material and the formation of bacterial biof iotalm.

Embodiment

Embodiment 1: leukodermic treatment

Vitiligo is a kind of acquired pigmentation disorder with dermal melanin cell impairment.Therefore, white speckle appears on the skin of body different parts.Vitiligo changes in the scope at 0.1-2% in global prevalence.It is destructive and can affect quality of life, self-respect, marriage and employment (Halder RM, Chappell JL.SeminCutan Med Surg.2009 that vitiligo can be psychology for influenced patient; 28 (2): 86-92.).Accurate pathogeny is still hard to understand, and the mechanism of proposition falls under autoimmune, biochemistry, oxidation antioxidation, nerve and viral title.Several studies show that for the free radical toxicity of melanocyte and accumulate the destruction that causes them.In fact, than control patients, the erythrocyte of patients with vitiligo has lower level glutathion.

In the present embodiment, we have reported that and two oxters all at her neck have leukodermic 45 years old women's of dispersivity case.Use following oral absorption sheet form be purchased Drug therapy she three months, therapeutic scheme: 0.6g NAC, three times/day; SeMet, 55 micrograms/once a day; Mel, 3mg/ is once a day; All these three kinds of medicines are three days/week equally, then interrupt 4 days (not administration).

At initial stage for the treatment of in three months with take the picture of her cervical region and two oxters latter stage.

Do not report adverse side effect.

From Fig. 2, the apparent decolouring of picture obviously reduces, and two oxters are secondary color almost completely.

Embodiment 2: beautifying skin is processed

Skin aging can be naked eyes identification, because there is gradually wrinkle, loss of elasticity, sagging and attenuation in adult.Therefore, measure skin surface texture is particular importance in dermatological field, because this type of mensuration can be used for skin diagnosis and treatment or cosmetic treatments assessment.

Environmental pollution has the dependency of increase for the impact of skin, relevant aging and malfunction is followed in often contaminated dose of degraded of skin.Skin is directly exposed to pollution, has and is evaluated as approximately 2 square metres surfaces, is greater than any other tissue.Owing to the damage mechanism of polluting mainly belong to induced oxidation stress and inflammatory reaction.These mechanism are ordered about the NAC of we use and SeMet and Mel combination in slightly aging, oily skin, the pimpled situation of tool.

One 43 years old women are because its oily skin, present redness and pimple and visit dermatologist.Adopt galenical to treat her one month, said preparation comprises: water, ceteareth 25 tristerins, triethanolamine stearate, cetearyl alcohol, NAC5%, SeMet0.5%, Mel0.1%, caprylic/capric triglyceride, simethicone, hydrogenating glycerine polyisobutylene, polysorbate 60, pentanediol, Rhizoma et radix valerianae.

The assessment of skin texture is on the silicone rubber skin copy of preparing dermatologist; then by microscopic examination (Fig. 3 A); follow the program of reporting (Setaro M, Sparavigna A.Irregularity skin index (ISI): a tool to evaluate skin surface texture.Skin Res Technol.2001; 7 (3): 159-63.).As what illustrated by these authors, by upper thread, to analyze, the microscopy of skin copy shows the change of skin surface texture systematicness.The cream preparation therapeutic alliance that employing comprises NAC, SeMet and Mel is after one month, face's upper thread (face surface line) performance is more regular, its network order has unforgettable increase, particularly when ining contrast to the front observed random coiling outward appearance for the treatment of.The image fast fourier transform (FFT) that the method for Setaro and Sparavigna followed is carried out is analyzed the true pattern before (Fig. 3 B) treatment with preferential direction-as by left hurdle A and B as shown in arrow, indicate irregular texture of showing.After treatment, fft analysis shows the substitute is the almost completely circular pattern of rule, indicates regular skin texture.

After the treatment moon, dermatologist and patient have reported that the irregular minimizing of visual skin is together with more compacting and more smooth outward appearance, smegma normalization and red and swollen and pimple disappearance.

Embodiment 3: for the antiopxidant effect of In vitro culture blastoid cell system (blastoid cell line)

Method

End user's lymphoblastoid (lymphoblastoid) HL60 and proerythroblast (proerythoblastoid) K562 cell line, be grown in RPMI1640 culture medium (Invitrogen at 37 ℃, Gibco, Milano, Italy) in, supplement 10% hyclone, 5 μ g/ml penicillins, 5 μ g/ml streptomycins, 2mM glutamine, and one week routine goes down to posterity and cultivates three times.With independent menadione (contrast), unique NAC, NAC+Se-methionine (SeMet), NAC+Mel or NAC+SeMet+Mel process cell.In the situation that supplementing SeMet, this replenishes test and a few days ago carries out, by using SeMet solution in buffer (concentration as Fig. 4-7 describe as shown in).In the situation that supplementing Mel, this replenishes to test and carries out for first 20 hours, by using alcoholic solution (concentration is as shown in the description of Fig. 4-7).Concentration of alcohol≤1.3% in growth medium and contrast only have this solvent also Parallel Growth.

By using fluorescent probe 5-(with-6)-chloromethyl-2'; 7'-dichloro-dihydro fluorescein diacetate; acetonyl ester (CM-H2DCFDA) (Invitrogen, Molecular Probes) detects NAC, Mel, SeMet and these combinations of substances Cell protections and avoids the effect that oxidation stimulates.CM-H2DCFDA is cell permeability active oxygen indicator, until remove its acetate groups by born of the same parents' lactonase, and just fluoresce at Cellular Oxidation.Thereby by fluorescence increase monitoring cell in hydroperoxides (be mainly H ₂o ₂) increase.By using menadione to carry out for increasing H ₂o ₂oxidation stimulate.

To cell counting, and note keeping ratios constant between cell and fluorescent probe.After cell washing, being suspended from concentration is 10 again ⁶in the 2ml HBSS of/ml, be then transferred to absorption cell and balance 15 minutes under gentle agitation at 37 ± 0.1 ℃ in exometer cell holder.Add 2 μ l CM-H2DCFDA DMSO solution and through time reading fluorescence intensity.At reading first after 200 seconds, add the NAC phosphate buffer (concentration as Fig. 4-7 describe as shown in) of suitable whole part, and the menadione alcoholic solution of 10 μ l0.1M, and continue reading fluorescence intensity.During intensity reading, sample is placed in and continues under gentle agitation.

In being furnished with the K2 exometer of xenon arc lamp (ISS Inc.Champaign, IL), continue fluorescence intensity 20 minutes, by using 495nm place to excite and the transmitting of 530nm place, band leads to for 8nm.

Result

The representative result of the Cellular Oxidation response after processing is separately shown in Fig. 4,5 and 6.The menadione of variable concentrations, NAC, SeMet and Mel are tested and provided different responses, and (response) also depends on cell line.Yet the combination of these three kinds of materials always causes the fluorescence that the most anti-oxidation is ordered about to increase.With menadione oxidation, challenging after (oxidative challenge), DCFDA fluorescence intensity increases.NAC adds the dynamic (dynamical) relevant minimizing of this strength increase causing as assessed by its slope.The SeMet or the Mel that together with NAC, exist have different effect, depend on cell type and concentration.

Apparently, observe in some cases NAC+SeMet or NAC+Mel to not being to reduce response but promote fluorescence to increase, hint enzymatic oxidation effect.One representative example is reported in Fig. 6.Yet, even if in these situations the combination of three kinds of composition NAC+SeMet+Mel also caused with respect to the slope of contrast and thereby the maximum of oxidation response reduce.

Powerful anti-oxidation protection by NAC+SeMet+Mel combinations can reduce 60%NAC concentration.This is shown in Fig. 7, and wherein the only 0.4mM NAC acquisition by use and SeMet and Mel combination equals to the protection of being brought into play by 1mM NAC.

Conclusion

The combination of NAC and SeMet and Mel has clearly increased the anti-oxidation protection of NAC.Cooperative effect has been brought into play in this combination, is not also for the effect of three kinds of components separately separately for NAC effect.In some situations, when NAC and SeMet and Mel are used in combination, antiopxidant effect is with respect to three times of independent NAC effectively.

Can reduce NAC concentration, use the concentration that is less than half to reach similar effect.

Unexpectedly, observe in some cases SeMet or Mel, also had the disadvantageous enzymatic oxidation effect with NAC combination.Although mechanism, still in arguement, has been reported the two anti-enzymatic oxidation effect (adverse pro-oxidant effect) of Se and Mel in the literature.Yet the combination of three kinds of materials in our test (wherein Se is the form of SeMet) has recovered antioxidation cytoprotective, even if also have the final effect of insignificant oxidation under oxidant challenge.

Embodiment 4: the differentiation effect in colon cancer cell.

Reported that before this NAC regulates cell proliferation and differentiation, particularly reduces propagation by transformant towards normal differentiation pathway.It is reported, this effect occurs in normal cell and cancerous cell.

Analyzed as follows the expression of propagation and differentiation associated gene in the colon carcinoma cell line (Caco-2) that the NAC of employing and SeMet or Mel or the two combination processes.The results are shown in Fig. 8 and 9.

Method

By colon cancer cell Caco-2 (sub-clone TC7, Chantret etc., J.Cell Sci.107:213 – 225; 1994) routine maintains in the high glucose DMEM (Gibco, Invitrogen, Milan, Italy) that comprises 10% heat-inactivated fetal bovine serum, FBS (Gibco, Invitrogen).On six orifice plates with 3x10 ³/ cm ²density inoculating cell.Inoculating after 24 hours adopts NAC (2mM) and/or SeMet (25nM) and/or Mel (25 μ M) to process cell.Process after 72 hours the gene expression of analysis of cells.

Assessment enterocyte breaks up peculiar gene expression; Two kinds of coding brush-border hydrolysis enzymes (brush border hydrolase), the gene of intestinal alkaline phosphatase (ALPI) and Sucrase-isomaltase alpha-Glucosidase (SI); Two kinds of Codocyte-cell adhesion complex albumen, tight junction protein 1 (tight junction claudin1) (CLDN1) and gene (PCDH1) of bonding protocadherin 1 (adherent junction protocadherin1).Also analyzed the important inhibitor of cell cycle, as the cyclin dependent kinase inhibitors 1A (CDKN1A/P21 that conventionally follows the cell cycle arrest label of Caco-2 cell differentiation ^waf1/cip1) expression.Use glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as house-keeping gene.

Use can be: http:// www.ncbi.nlm.nih.govthe software Primer-BLAST design specific primer sequence obtaining is also summarised in table 1.

In TRIzol reagent (Invitrogen), extract total RNA.Adopt 1U DNAse I (Invitrogen) processing from the total RNA of a microgram of each sample and adopt 200U SuperScript III reverse transcriptase (Invitrogen) reverse transcription, in 20 μ l (final volume), using 250ng random primer.At ABI Prism7000 (Applied Biosystems, Monza, Italy) on, carry out real-time quantitative, in the 25 μ l reaction volumes that comprise 10ng cDNA and 0.6 each primer of μ l (10 μ M), use 5Prime RealMasterMix SYBR ROX2.5X (Eppendorf, Milan, Italy), according to following scheme: circulation in 10 minutes 1 at 95 ℃, three steps 40 circulations, comprise at 95 ℃ 20 seconds, at 60 ℃ 40 seconds, and at 68 ℃ 45 seconds.All tests repeat three times or more times is analyzed separately.Each reaction operation negative control (without template) is pollution-free to confirm, and amplifies specificity by melting curve analysis confirmation.All data analysiss are to have the meansigma methods of standard deviation and use ANOVA to pass through p parameter evaluation significance.For measuring relative transcript abundance, use 2 ^{-Δ Δ CT}method (Livak KJ, Schmittgen TD.Methods2001; 25:402-408.).

Table 1

Result

The results are shown in Fig. 8 and 9, shown that the gene expression that is placed in five kinds of genes while processing separately when cell increases (multiple variation).Real-time quantitative RT-PCR data show without doubt:

1), than independent NAC, by combining with SeMet and Mel, the antiproliferative effect of NAC (as shown in expressing as increased by P21 gene) doubles.

2) differentiation of NAC (expressing indicated as increased by marker gene SI, ALPI, CLDN1, PCDH1) has clearly strengthened by above combination, and the expression of marker gene has increased 40-80% than independent NAC.

3) although when analyzing some specific differentiation marker things, the combinations of pairs of for example observing NAC+SeMet or NAC+Mel when ALPI, CLDN1 and PCDH1 has the effect that equals to three kinds of combinations of substances.Yet when being used, three kinds of combinations of substances there is the whole trend of knowing that increases induction.

Embodiment 5: the antimicrobial effect of NAC, SeMet and Mel in medical treatment device hydrogel coating

Method

Hydrogel coating:

Use as be known in the artly based on polyurethane (PUR) and polyvinylpyrrolidone (PVP), be designed for the water-insoluble hydrogel polymer coating of medical polymer devices, apply polrvinyl chloride catheter and latex catheter.Before the sign of this hydrogel coating by Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectrum, carry out with respect to uncoated framework material with to Static and dynamic friction factor, water-wet angle and the microexamination of porcine tissue counterface (porcine tissue counter-face).The test that inventor carries out (data are not shown) confirms the change of hydrated state lower surface composition, Superhydrophilic and lubricity, comprises that friction factor reduces.In thering is the polrvinyl chloride catheter situation of hydrogel coat internal surfaces, observe capillarity phenomenon, proved the high-affinity between coating and hydrone.

Bacterial growth and biofilm formation:

For measuring the impact forming for adhering to set antibacterial (adherent sessile bacteria), according to (the Antimicrob Agents Chemother.1985 of the technique described in Nickel and companion; 27 (4): 619-24) build artificial conduit.The tube material that uses is latex, adopt the latex that applies containing noble metal hydrogel, adopt the latex that NAC, SeMet and Mel (NAC+SeMet+Mel) apply and adopt noble metal and (NAC+SeMet+Mel) latex of the two coating.Artificial pipe guide is connected to two liters of containers, and it is as remaining on the external bladder in 37 ℃ of water-baths.The pump of sending 50ml/h by being made as will pass through this artificial conduit from container pumping containing the culture medium of escherichia coli (E.coli).Before being exposed to escherichia coli and test afterwards this conduit 10cm ²position, stage casing.The separated patient from thering is conduit dependency urinary tract infection of coli strain who uses in these trials.Used medium is the artificial urine that supplements 0.4% nutrient broth.This antibacterial is stored at-70 ℃ on the inclination agar in test tube (inclination) and with 10 hours interval continuous culture.By adopting spectrophotometer (600nm) to be used as the standardization turbidity of growth parameter(s) to monitor this external intravesical bacterial growth.

Make containing colibacillary artificial urine by artificial conduit 10 hours and by the development of sampling catheter material surface monitoring bacterial biof iotalm.The aseptic sample disk that removes carrying set bacterium is for scanning electron microscopy.The surface of in addition, scraping catheter tray by low output sound wave in sterile phosphate buffer salt solution obtains the quantitative counting there is vigor particle-bound bacteria.Arrange dilution progression to 10 ^-4and spread upon on Nutrient agar, therefrom obtain quantifying board counting.From artificial conduit, remove appointment for the catheter specimen of SEM and be placed on the dimethyl arsenic acid buffer liquid (0.1M by 5% glutaraldehyde, pH7.2) in the fixative forming at 22 ℃, 1 hour, then in a series of aqueous alcohol solutions, (in 20-100% and Freon113-alcoholic solution (30-100%), dewater and then dry.In gilding machine, with gold, apply sample and pass through to use scanning electronic microscope examination.

Result

The growth of catheter tray surface scrape on Nutrient agar shows: than uncoated framework material, the vigor escherichia coli number that has on hydrogel coating material significantly reduces.When being added to hydrogel, NAC, SeMet and Mel be concerned about the further rapid reduction of particle-bound bacteria clump count object.

Bacterium colony number on framework material: 100

Bacterium colony number on hydrogel coating material: 73

There is the bacterium colony number on the hydrogel coating material of NAC, SeMet and Mel: 58

SEM check to show that conduit latex, noble-metal coated latex, (NAC+SeMet+Mel) apply latex and noble metal+(NAC+SeMet+Mel) and apply the ethanol that latex dish do not have significant quantity before being exposed to escherichia coli and kill bacterial cell.Be exposed in artificial urine Bacillus coli cells after 10 minutes, on latex dish, see the adhesion bacterial cell of significant quantity and on coating disk, there is minimum bacterium colony at noble-metal coated latex or (NAC+SeMet+Mel), and on the coating disk of noble metal+(NAC+SeMet+Mel) without bacterium colony.After field planting 10 hours, the characteristic lamellar surface of uncoated latex dish is adhered in a large number antibacterial and is entirely shut, and its amorphous extracellular polysaccharide that is embedded into a large amount of antibacterials self is to form thick attached biological film.At noble-metal coated catheter tray and (NAC+SeMet+Mel) apply on catheter tray, there is rare biofilm formation phenomenon and on the coating disk that adopts noble metal and combination (NAC+SeMet+Mel) inanimate object film.

Claims (20)

1. medical product, comprises separately or together

(i) ACETYLCYSTEINE;

(ii) selenium of selenomethionine form; With

(iii) melatonin

And/or the upper acceptable salt of its physiology.

2. according to the medical product of claim 1, for simultaneously, order or separately combination medicine-feeding to mammal.

3. according to the medical product of claim 2, component (i) and (ii) with (iii) for oral administration wherein.

4. according to the medical product of claim 3, wherein component (i) is the dosed administration with 5～45mg/kg/ days, and component (ii) is to be the dosed administrations with 0.02～0.08mg/kg/ days with dosed administration and the component (iii) of 0.4～1.2 μ g/kg/ days.

According to before the medical product of any one in claim, wherein this medical product is to comprise component (i) N-acetyl-cysteine (ii) selenomethionine and (iii) pharmaceutical composition of melatonin.

6. according to the medical product of claim 1 or 2, component (i) N-acetyl-cysteine wherein, (ii) selenomethionine and (iii) melatonin be for epithelium administration.

7. according to the medical product of claim 6, wherein component (i) is the concentration administration with 3～10wt-%, and component (ii) is to be the concentration administration with 0.01～0.2wt-% with the concentration administration of 0.3～1wt-% and component (iii).

According to before the medical product of any one in claim, be used for the treatment of optimum or malignant tumor and form.

9. according to the medical product of claim 1-7, be used for the treatment of dermatosis.

10. according to the medical product of claim 1-7, for beautifying skin, process.

11. according to the medical product of claim 1, as the antibacterial of the medical treatment device that is applicable to contact with body fluid.

12. according to the medical product of claim 11, wherein component (i) ACETYLCYSTEINE, (ii) selenomethionine and (iii) melatonin be contained in the hydrogel coating for medical treatment device.

13. according to the medical product of claim 11-12, further comprises additional activity compound for example germ killing drugs, precious metal solution and/or dexamethasone.

14. antibacterial that comprise claim 1 medical product.

15. according to the antibacterial of claim 14, and for hydrogel coating, this hydrogel coating is for medical treatment device.

16. according to the antibacterial of claim 14, for the plastic concordancy with medical treatment device.

17. medical treatment devices, the medical product that it comprises claim 1 and optional additional activity compound be germ killing drugs, precious metal solution and/or dexamethasone for example.

18. according to the purposes of the medical product of claim 1-7, is used for the treatment of and comprises that the optimum of various cancer types and malignant tumor formation, autoimmune disease, neurodegenerative diseases, endocrinopathy, type 2 diabetes mellitus, all types of fibrosis, amyloidosis, endometriosis, polycystic ovarian syndrome, dysmenorrhea, dermatosis comprise that vitiligo, alopecia or psoriasis or antibacterial infect.

19. according to the purposes of the medical product of claim 1-7, for beautifying skin, processes.

20. according to the purposes of the medical product of claim 1, for the antibacterial of the medical treatment device as being applicable to contact with body fluid.

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